The process of introducing desirable wild-type traits, such as disease resistance, plant habit and the like, into cultivated varieties has long been a basis for improving crop plants. Sexual hybridization is the traditional method used in crop improvement, but its use may be limited because of the natural barriers to hybridization which frequently exist in cultivated species. This particular effort has been severely restricted in the cultivated tomato Lycopersicon esculentum, because the opportunity for gene flow between the cultivated variety and its wild relatives is so curtailed by interspecific incompatabilities. Alternative methods to traditional breeding schemes, such as protoplast fusion and regeneration, have also been intensely investigated in tomato; however, these techniques as applied have had very limited success since the tomato, especially the cultivated variety, is much more resistant to regeneration from protoplasts than are other solanaceous species such as tobacco or potato.
In an attempt to overcome the difficulty of regenerating tomato, those interested in producing somatic hybrids of tomato have fused tomato protoplasts with protoplasts of certain species of the genus Solanum, which is more commonly easily regenerable. Although this technique frequently does allow the regeneration of protoplast fusion derived plants, a disadvantage arises in that the regeneration does require the expression of genes from Solanum. Since evidence suggests that genes controlling regeneration are polygenic and unlinked, all previous selection systems have produced regenerated plants which would necessarily have a minimum of several Solanum chromosomes.
Because previous systems have generally relied on Solanum species for regeneration, the known reports of somatic hybridization have nearly all been between two distinct genera, Lycopersicon and Solanum. As might be expected, however, because of the taxonomic distance between the fusion partners, the resultant plants are almost always completely infertile. For example, Melchers, et al. (Carlsberg Res. Commun. 43: 203-18, 1978) described fusion products between L. esculentum and the potato, S. tuberosum; however, all resulting plants were completely sterile and therefore not useful in a breeding program. This result was later confirmed by Shepard, et al. (Science 219: 683-688, 1983). Similarly, Jain, et al. (Abst. First Int. Cong. Plant . Mol. Biol., p.61, 1985) in fusing protoplasts of L. esculentum and S. nigrum recovered a number of somatic hybrids, but none of the regenerated plants were fertile.
In an attempt to use Solanum-Lycopersicon hybrids as breeding material, Handley, et al. Theoret. Appl. Genet. 71: 691-697, 1986) devised a method of protoplast fusion between Solanum lycopersicoides and Lycopersicon esculentum, in which the selection for hybrids at least nominally favored the L. esculentum parent. However, although in principle the tomato was favored, the selection step which relies on speed of regeneration actually favors S. lycopersicoides, which grows faster than L. esculentum. Therefore, ultimately, the Handley method uses a medium which in fact favors Solanum over Lycopersicon.
An attempt has also been made to fuse L. esculentum with the more closely related wild species, L. pennellii (M. O'Connell, et al., Theoret. Appl. Genet. 70: 1-12, 1985). Although the sexual hybrid of these two species grows well in culture and is readily regenerable from protoplasts, the somatic hybrids produced only callus with no hybrid plants ultimately produced.
The present invention, on the other hand, relates to a method of producing tomato somatic hybrids and cybrids in which the foregoing problems are eliminated. As used throughout the specification and claims, the term "hybrid" is intended to encompass both somatic hybrids and cybrids. The method utilizes a medium which truly selects for L. esculentum so that only unfused L. esculentum cells, L. esculentum cybrids with L. esculentum nuclear genes, and somatic hybrids between L. esculentum and related wild species are regenerated. Thus, the present method is more likely to ultimately yield gene combinations which contain a full tomato genome than previous methods have been.